The long-term objective of the proposed research is to gain an understanding of the molecular mechanism of visual excitation in vertebrate retinal rods. Many recent studies have suggested that cGMP is involved in the visual transduction process. The change in cGMP concentration in the rod outer segments is regulated by a light-activated cGMP phosphodiesterase and a GTPase. The specific aim of my proposed study is to determine how the activities of these two enzymes are controlled in the rods. I proposed to carry out the following biochemical studies: (1) The role of the rhodopsin and guanyl nucleotides in phosphodiesterase and GTPase activation will be investigated by a novel equilibrium transfer method that can separate photolyzed rhodopsin from the activated phosphodiesterase and GTPase in a membrane suspension. (2) The interaction between the phosphodiesterase and the GTPase in solution will be determined by equilibrium centrifugation and chemical cross-linking. Recombination and binding experiments using purified GTPase, phosphodiesterase, and reconstituted membranes will be carried out. The effect of guanyl nucleotides in promoting effective reconstitution of the light-regulated enzymatic activities will be studied. (3) The regions of the rhodopsin molecule that are essential for GTPase and PDE activation will be analyzed by chemical modification, phosphorylation, and proteolysis. (4) The lipid composition and physical state of the disk membranes will be modulated by cholesterol enrichment in order to determine the role of the membrane lipids in GTPase and phosphodiesterase activation. The effect of lipid transition and rhodopsin aggregation on the photoamplification process will be investigated by using a recombinant of the two enzymes and reconstituted membranes containing synthetic lipids.